WO2024071043A1 - Procédé de production de protéine hétéromultimère, protéine, acide nucléique, vecteur d'expression, transformant et procédé de production de protéine - Google Patents

Procédé de production de protéine hétéromultimère, protéine, acide nucléique, vecteur d'expression, transformant et procédé de production de protéine Download PDF

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WO2024071043A1
WO2024071043A1 PCT/JP2023/034748 JP2023034748W WO2024071043A1 WO 2024071043 A1 WO2024071043 A1 WO 2024071043A1 JP 2023034748 W JP2023034748 W JP 2023034748W WO 2024071043 A1 WO2024071043 A1 WO 2024071043A1
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binding
domain
protein
tag
cleavage
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幸樹 真壁
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国立大学法人山形大学
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the present disclosure relates to a method for producing a heteromultimeric protein, a protein, a nucleic acid, an expression vector, a transformant, and a method for producing a protein.
  • a single antibody molecule has antigen-binding domains that exhibit binding affinity to different antigens, and by changing the target antigen, development is underway for a variety of medical applications, such as anticancer drugs and hemophilia treatment drugs.
  • a bispecific antibody is composed of two types of heavy chains (H chains) and two types of light chains (L chains). Furthermore, when two types of H chains and two types of L chains are expressed to produce the bispecific antibody, there are 10 possible combinations of the two H chains and two L chains in the expressed antibody, which means that in addition to the desired bispecific antibody, as many as nine types of unnecessary antibodies are produced (Patent Document 1).
  • the present disclosure therefore aims to provide a new method for producing heteromultimeric proteins such as bispecific antibodies.
  • the production method of the present disclosure is a method for producing a heteromultimeric protein, comprising the steps of: A complex formation step in which two proteins are contacted to form a first complex of the two proteins,
  • the two proteins include a first protein and a second protein, the first protein comprises, in order from N-terminus to C-terminus, a first binding tag, a first cleavage domain, and a first domain;
  • the second protein comprises, in order from N-terminus to C-terminus, a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain;
  • the first protein and the second protein are capable of forming a dimer by binding between the first domain and the second domain; forming the first complex by binding between the first binding tag and the first binding partner and between the first domain and the second domain; and a generating step of cleaving the first cleavage domain and the second cleavage domain in the first complex to generate
  • the protein of the present disclosure includes, from the N-terminus to the C-terminus, a first binding tag capable of binding to a first binding partner, a first cleavage domain, and a first domain, in that order.
  • the protein of the present disclosure includes, from the N-terminus to the C-terminus, a first binding partner capable of binding to a first binding tag, a second cleavage domain, and a second domain, in that order.
  • the protein of the present disclosure includes, from the N-terminus to the C-terminus, a second binding tag capable of binding to a second binding partner, a third cleavage domain, and a third domain, in that order.
  • the protein of the present disclosure includes, from the N-terminus to the C-terminus, a first binding partner capable of binding to a second binding tag, a fourth cleavage domain, and a fourth domain, in that order.
  • the proteins of the present disclosure include two proteins:
  • the two proteins include a first protein and a second protein, the first protein comprises, in order from N-terminus to C-terminus, a first binding tag, a first cleavage domain, and a first domain;
  • the second protein comprises, in order from N-terminus to C-terminus, a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain;
  • the first protein and the second protein form a dimer by binding between the first domain and the second domain;
  • the first binding tag and the first binding partner are linked.
  • the nucleic acid of the present disclosure encodes the protein of the present disclosure.
  • the vector of the present disclosure contains the nucleic acid of the present disclosure.
  • the transformant of the present disclosure contains the nucleic acid and/or vector of the present disclosure.
  • the method for producing the protein of the present disclosure includes an expression step for expressing the nucleic acid and/or vector of the present disclosure.
  • the present disclosure provides a new method for producing heteromultimeric proteins such as bispecific antibodies.
  • FIG. 1 is a schematic diagram showing an example of protein components used in the production method of the present disclosure and each step of the production of a heterodimeric protein.
  • FIG. 2 is a schematic diagram showing an example of protein components used in the production method of the present disclosure and each step of the production of a heterotetrameric protein.
  • FIG. 3 is a schematic diagram showing an example of protein components used in the production method of the present disclosure and each step of the production of a heterotetrameric protein.
  • FIG. 4 is a photograph showing the results of SDS-PAGE.
  • FIG. 5 is a photograph showing the results of SDS-PAGE.
  • FIG. 6 is a graph showing the elution pattern of Herceptin by size exclusion chromatography.
  • FIG. 1 is a schematic diagram showing an example of protein components used in the production method of the present disclosure and each step of the production of a heterodimeric protein.
  • FIG. 2 is a schematic diagram showing an example of protein components used in the production method of the present disclosure and each step
  • FIG. 7 is a graph showing binding of the light chain of the CD3 antibody and the heavy chain of the CD3 antibody to CD3 positive cells by flow cytometry.
  • FIG. 8 is a graph showing binding of the Herceptin antibody light chain and the Herceptin antibody heavy chain to HER2 positive cells by flow cytometry.
  • FIG. 9 is a photograph showing the results of SDS-PAGE.
  • FIG. 10 is a photograph showing the results of SDS-PAGE.
  • a "protein” refers to a polymer of peptides composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids.
  • the polymer may be, for example, linear, branched, or cyclic.
  • the protein may also be referred to as a peptide or a polypeptide.
  • monomeric protein refers to a protein that is not bound to or associated with other proteins.
  • dimeric protein refers to a protein complex in which two proteins or protein subunits are bound or associated. When the two proteins are the same protein or subunit, the dimeric protein can also be called a homodimeric protein. When the two or more proteins are different proteins or subunits, the dimeric protein can also be called a heterodimeric protein.
  • tetrameric protein refers to a protein complex in which four proteins or protein subunits are bound or associated. When the four proteins are the same protein or subunit, the tetrameric protein can also be called a homotetrameric protein. When one or more of the four proteins or subunits are different proteins or subunits from the others, the tetrameric protein can also be called a heterotetrameric protein.
  • multimeric protein refers to a protein complex in which two or more proteins or protein subunits are bound or associated with each other.
  • binding tag refers to a polypeptide or substance that has specific binding properties with another molecule.
  • binding partner refers to a polypeptide or substance that has specific binding affinity to the binding tag.
  • domain refers to a structurally or functionally integrated region of a “protein,” “polypeptide,” and/or “peptide.”
  • cleavage domain refers to a domain composed of a peptide that is cleaved or decomposed into two or more regions by a substance having self-cleavage activity or other cleavage activity.
  • substances having other cleavage activity include proteases, peptidases, modified inteins, etc.
  • antibody refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • Immunoglobulin genes include genes encoding constant regions such as ⁇ , ⁇ , ⁇ (including ⁇ 1 and ⁇ 2), ⁇ (including ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), ⁇ , ⁇ , and ⁇ , and genes capable of encoding countless immunoglobulin variable regions such as V regions, D regions, and J regions.
  • the antibody comprises, for example, a heavy chain and a light chain.
  • the light chain comprises ⁇ and ⁇ , constituting the ⁇ chain and the ⁇ chain, respectively.
  • the heavy chain comprises ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , constituting the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.
  • the antibody may be a typical immunoglobulin (antibody) structural unit composed of a tetramer.
  • the antibody is composed of two identical pairs of polypeptide chains, each pair consisting of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa).
  • the N-terminus of each chain also defines a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition.
  • antigen-binding fragment refers to a portion or partial polypeptide that comprises the antigen-binding site of an antibody.
  • the antigen-binding fragment can be obtained by chemical or enzymatic treatment of an antibody.
  • the antigen-binding fragment can also be obtained by recombinant means.
  • the antigen-binding fragment can be, for example, Fab, Fab', F(ab') 2 , Fc, and/or Fv fragments, and derivatives thereof.
  • purification means identifying, separating, recovering from a component in its natural state, being identified and separated, and/or being recovered from a component in its natural state.
  • the “purification” can be performed, for example, by obtaining at least one purification step.
  • the purification can also be referred to as isolation.
  • separation means separating a target object from a substance that contains said target object and/or the state of being separated. Said separation can also be referred to as liberation.
  • nucleic acid refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • nucleic acid refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA.
  • the nucleic acid may be, for example, single-stranded or double-stranded.
  • the nucleic acid may be interchangeably referred to as a "polynucleotide” or a "nucleic acid molecule.”
  • host refers to a cell and/or an individual into which exogenous nucleic acid is introduced.
  • the host can also be referred to as a host cell.
  • vector and “expression vector” refer to a recombinant plasmid or virus that contains a nucleic acid to be delivered to a host or host cell in vitro or in vivo .
  • the "vector” and “expression vector” include viral vectors and non-viral vectors.
  • transformant refers to a host into which foreign nucleic acid has been introduced.
  • each protein, polypeptide, or peptide is not particularly limited and may be any animal.
  • the animal may be, for example, a human or a non-human animal.
  • the non-human animal may be, for example, a mammal such as a mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, dolphin, or sea lion.
  • the present disclosure provides a method for producing a heteromultimeric protein, the method comprising a complex formation step of contacting two proteins to form a first complex of the two proteins, the step comprising:
  • the two proteins include a first protein and a second protein, the first protein comprises, in order from N-terminus to C-terminus, a first binding tag, a first cleavage domain, and a first domain;
  • the second protein comprises, in order from N-terminus to C-terminus, a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain;
  • the first protein and the second protein are capable of forming a dimer by binding between the first domain and the second domain; forming the first complex by binding between the first binding tag and the first binding partner and between the first domain and the second domain; and a generating step of cleaving the first cleavage domain and the second cleavage domain in the first complex to generate a heterodimer
  • the inventors came up with the idea that it might be possible to efficiently produce a dimer of two proteins by adding a specific binding tag or binding partner to each of the proteins that form a dimer and forming a complex through binding between the binding tag and the binding partner.
  • a heterodimeric protein can be obtained by cleaving the cleavage domain after the complex is formed, and thus established the present disclosure.
  • the estimated reaction mechanism of the production method of the present disclosure will be explained using an example of producing a heterodimeric protein, but as described below, the production method of the present disclosure can also be applied to the production of proteins other than heterodimeric proteins. Therefore, the production method of the present disclosure can produce heteromultimeric proteins.
  • the first protein 1 contains a first binding tag 11, a first cleavage domain 12, and a first domain 13, in this order from the N-terminus to the C-terminus.
  • the second protein 2 contains a first binding partner 21 capable of binding to the first binding tag 11, a second cleavage domain 22, and a second domain 23, in this order from the N-terminus to the C-terminus.
  • the first protein 1 and the second protein 2 are brought into contact with each other. As a result, a bond is formed between the first domain 13 and the second domain 23, as indicated by X. Also, as indicated by the arrow Y, a bond is formed between the first binding tag 11 and the first binding partner 21. As a result, the first protein 1 and the second protein 2 form a first complex 10.
  • the first cleavage domain 12 and the second cleavage domain 22 of the first complex 10 are cleaved. As a result, the first binding tag 11 and the first binding partner 21, which are located on the N-terminal side of the first cleavage domain 12 and the second cleavage domain 22, are detached from the first complex 10.
  • a heterodimeric protein 20 including the first domain 13 and the second domain 23 can be produced. Therefore, it is presumed that the manufacturing method of the present disclosure can produce a desired heterodimer protein by placing monomer proteins that constitute a desired heterodimer in the first domain 13 and the second domain 23.
  • the first complex is formed by binding between the first binding tag and the first binding partner, and binding between the first domain and the second domain.
  • the first protein and the second protein can be prepared by genetic engineering techniques, for example, as described later in the protein manufacturing method of the present disclosure. Therefore, the manufacturing method of the present disclosure may optionally include a first expression step of expressing the first protein and the second protein in a host cell prior to the complex formation step.
  • the expression method in the expression step can be based on the explanation of the protein, nucleic acid, expression vector, transformant, and protein manufacturing method of the present disclosure described later.
  • the first protein is reacted with the second protein.
  • the first binding tag and the first binding partner bind, and the first domain binds to the second domain, forming a first complex between the first protein and the second protein.
  • the complexation between the first protein and the second protein may be, for example, (1) caused by binding between the first binding tag and the first binding partner, (2) caused by binding between the first domain and the second domain, or caused by binding between both (1) and (2).
  • the complexation is caused by binding between both (1) and (2) because this can improve the dimer formation ability.
  • the bond between the first domain and the second domain may be a direct bond, an indirect bond (association), or may be formed by both a direct bond and an indirect bond between the first domain and the second domain, but is preferably a direct bond.
  • the direct bond is a covalent bond, and specific examples thereof include an amide bond (peptide bond, isopeptide bond, etc.) between amino acids, a disulfide bond between cysteines, etc.
  • the indirect bond is a non-covalent bond, and specific examples thereof include a hydrogen bond, a hydrophobic bond, etc.
  • the first domain and the second domain can adopt an amino acid sequence capable of forming a dimer, condition-dependent or condition-independent, when proteins containing each domain coexist.
  • the first domain and the second domain can utilize, for example, an amino acid sequence of a motif sequence that forms each subunit in a protein dimer or a dimer thereof.
  • the first domain and the second domain can utilize an amino acid sequence of a motif sequence that forms each subunit in a protein multimer or a dimer thereof.
  • the protein dimer can be a homodimer or a heterodimer.
  • protein dimer examples include heavy and light chain dimers of immunoglobulins (antibodies) such as IgA, IgD, IgE, IgG, and IgM; proteins containing leucine zippers such as AP-1 (c-fos and c-jun), myc family proteins such as myc, max, and mdx1; G protein-coupled receptors; kinesin; receptor tyrosine kinases such as the ErbB receptor family, platelet-derived growth factor receptor (PDGFR), neurotrophin (neurotrophic factor) receptors, insulin receptors, insulin-like growth factor receptors, vascular endothelial growth factor receptors (VEGFR), and stem cell factor receptors; and Toll-like receptors such as TLR1 to 11.
  • immunoglobulins immunoglobulins
  • proteins containing leucine zippers such as AP-1 (c-fos and c-jun), myc family proteins such as myc, max, and mdx1
  • the first domain and the second domain include, for example, the light chain and the heavy chain of an antibody that binds to a first target antigen, respectively.
  • the antibody is, for example, IgA, IgD, IgE, IgG, or IgM, and is preferably IgG.
  • the IgG is, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4.
  • the antibody is, for example, an animal-derived antibody, and specific examples include a human antibody, a mouse antibody, a chicken antibody, a rat antibody, and a rabbit antibody.
  • the first domain and the second domain are preferably human-derived antibodies, and more preferably human-derived IgG.
  • amino acid sequences of human IgG1, IgG2, IgG3, and IgG4 can be, for example, the amino acid sequences registered in UniProt under accession numbers P01857, P01859, P01860, and P01861, respectively.
  • the antibody may be an antibody with a modified constant region.
  • the first domain and the second domain may include a light chain or heavy chain amino acid sequence containing a modified constant region.
  • modified constant region examples include Fcab (Fc antigen binding, References 1 and 5) in which the amino acid sequence of the constant region is modified to give it the ability to bind to a target molecule, IgG hexamer (References 2 to 4) in which the amino acid sequence of the constant region of an IgG antibody is modified to give it the ability to form a hexamer, DAF (Dual Action Fab, Reference 5), Charge pair (Amgen, Reference 5), SEEDbody (Reference 5), Knobs-in-holes (Reference 5), DVI-IgG (Reference 5), and the like.
  • Fcab Fc antigen binding, References 1 and 5
  • IgG hexamer References 2 to 4
  • amino acid sequence of the constant region of an IgG antibody is modified to give it the ability to form a hexamer
  • DAF Dual Action Fab, Reference 5
  • Charge pair Amgen, Reference 5
  • SEEDbody Reference 5
  • Knobs-in-holes Reference 5
  • Reference 3 de Jong RN et al., “A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.”, PLoS. Biol. (2016) 14(1): e1002344.
  • Reference 4 Christoph A. Diebolder et al., “Complement Is Activated by IgG Hexamers Assembled at the Cell Surface”, Science, 343 (6176), pages 1260-1263
  • Reference 5 Christoph Spiess et al., “Alternative molecular formats and therapeutic applications for bispecific antibodies”, Molecular Immunology, Volume 67, Issue 2, Part A, 2015, Pages 95-106
  • the first binding tag and the first binding partner are molecules that bind to the first binding partner condition-dependently or condition-independently when a protein containing the first binding tag and a protein containing the first binding partner coexist.
  • the binding between the first binding tag and the first binding partner may be direct or indirect.
  • the first binding tag and the first binding partner can be, for example, a peptide tag and a peptide capable of spontaneously forming a covalent bond, or a peptide tag and a peptide capable of forming a covalent bond due to the modifying activity of another molecule.
  • examples of the first binding tag and the first binding partner include a Streptococcus pyogenes surface protein (SpyCatcher, SEQ ID NO: 1) and a peptide tag capable of binding to the SpyCatcher (SpyTag, SEQ ID NO: 2) or a variant thereof; a Streptococcus pneumoniae protein (SnoopCatcher, SEQ ID NO: 3) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag, SEQ ID NO: 4) or a variant thereof; a modified Clostridium perfringens protein Cpe0147 439-563 (SEQ ID NO: 5) and a peptide tag Cpe0147 565-587 (SEQ ID NO: 6) capable of binding to the Cpe0147 439-563 or a variant thereof; and the like.
  • SpyCatcher Streptococcus pyogenes surface protein
  • SpyTag SEQ ID NO: 2
  • SpyTag Streptococc
  • SpyCatcher and SpyTag examples include SpyCatcher2 and SpyTag2 (Reference 6), SpyCatcher3 and SpyTag3 (Reference 7), SnoopCatcher and SnoopTag (Reference 8), etc.
  • These peptide tags and peptides capable of spontaneously forming a covalent bond are bound by, for example, an isopeptide bond.
  • Keeble et al. “Approaching infinite affinity through engineering of peptide-protein interaction”, PNAS, 2019, vol. 116, No. 52, pages 26523-26533 Reference 8: Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M. Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):1202-7. doi: 10.1073/pnas.1519214113.
  • SpyCatcher Amino acid sequence of Streptococcus pyogenes surface protein (SpyCatcher) (SEQ ID NO:1) DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVN Amino acid sequence of SpyTag (SEQ ID NO:2) AHIVMVDAYKPTK
  • SnoopCatcher Amino acid sequence of Streptococcus pneumoniae protein (SnoopCatcher) (SEQ ID NO:3) KPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNDKHYITNEPIPPK Amino acid sequence of SnoopTag (SEQ ID NO:4) KLGDIEFIKVNK
  • the first binding tag and the first binding partner can be, for example, a K tag and a Q tag.
  • the K tag and the Q tag can form a covalent bond by crosslinking the N-terminal lysine residue of the K tag with the N-terminal glutamic acid of the Q tag using, for example, bacterial transglutaminase.
  • the first binding tag and the first binding partner can be, for example, an affinity tag and a molecule that binds to the affinity tag.
  • the first binding tag and the first binding partner can be, for example, a peptide, a polypeptide, or a protein.
  • binding tag examples include His-tag (Hisx6), His-Strep-tag, strep-tag, avidin tag, flag (trademark)-tag, HA (hemagglutinin)-tag, T7-tag, V5-peptide-tag, GST (glutathione-S-transferase)-tag, CBP (calmodulin-binding peptide)-tag, MBP (maltose-binding protein)-tag, Myc-tag, etc.
  • the binding tag may be a peptide consisting of any amino acid sequence to which the molecule exhibiting specific binding can bind.
  • the first binding partner can be appropriately set depending on the type of the first binding tag.
  • Specific examples of the first binding partner include an antibody or an antigen-binding fragment thereof that recognizes the first binding tag, or a derivative thereof; a nucleic acid molecule such as an aptamer; glutathione, calmodulin; a sugar chain such as mannose; a metal such as nickel, cobalt, or zinc, or an ion thereof; and the like.
  • the combination of the first binding tag and the first binding partner may be any combination that allows the first binding tag and the first binding partner to bind to each other.
  • the first binding partner when the first binding tag includes a His-tag, the first binding partner may be, for example, nickel.
  • the first binding tag includes a strep-tag or an avidin tag the first binding partner may be, for example, biotin.
  • the first binding tag includes an epitope tag such as a flag (trademark)-tag, an HA-tag, a T7-tag, a V5-peptide-tag, and/or a Myc-tag
  • the first binding partner may be, for example, an antibody against each epitope tag or an antigen-binding fragment thereof, or a derivative thereof.
  • the first binding partner may be, for example, glutathione.
  • the first binding partner may be, for example, calmodulin.
  • the first binding tag includes an MBP tag, the first binding partner can be, for example, mannose.
  • the first binding tag and the first binding partner may be functional equivalents to the extent that they maintain the binding ability between the first binding tag and the first binding partner.
  • the functional equivalent may be, for example, an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the reference amino acid sequence of the first binding tag or the first binding partner, and a polypeptide having binding ability with the corresponding first binding tag or first binding partner.
  • the functional equivalent may be, for example, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and/or added in the reference amino acid sequence of the first binding tag and the first binding partner, and a polypeptide having binding ability with the corresponding binding tag or binding partner.
  • the one or several are, for example, 1 to 44, 1 to 33, 1 to 22, 1 to 11, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1.
  • the substitution is preferably, for example, a conservative substitution.
  • the first protein may have one or more first binding tags.
  • the first binding tags may be of one type or of multiple types.
  • the first binding tag is located on the N-terminus of the first protein.
  • the second protein may have one or more first binding partners.
  • the first binding partner may be of one type or of multiple types.
  • the first binding partner is preferably located on the N-terminus of the second protein.
  • the number of the first binding tags and the first binding partners in each protein may be the same or different, but is preferably the same.
  • the first binding tag and the first binding partner are interchangeable, and the combinations described above may be used interchangeably.
  • the first cleavage domain and the second cleavage domain are domains having amino acid sequences that cause domain cleavage in a condition-dependent or condition-independent manner.
  • the cleavage is carried out in the production step described below after the formation of the first complex.
  • the first cleavage domain and the second cleavage domain are preferably cleavage domains that cause cleavage in a condition-dependent manner.
  • the first cleavage domain and the second cleavage domain may be the same cleavage domain or different cleavage domains. Furthermore, the first cleavage domain and the second cleavage domain may be one or more. In the latter case, the cleavage domain may be one type or more types.
  • both cleavage domains can be cleaved in a single reaction in the production process described below, and therefore a heterodimer protein can be produced efficiently.
  • the first cleavage domain and/or the second cleavage domain preferably contain a self-cleaving peptide and/or a cleavage sequence of a protease or peptidase.
  • the self-cleaving peptide include a 2A self-cleaving peptide, and an intein or a modified form thereof.
  • the protease or peptidase cleavage sequence include a thrombin cleavage sequence, a Factor Xa recognition sequence, a GST fusion protein cleaving enzyme (PreScissionTM Protease) recognition sequence, a furin-sensitive sequence, and a carboxypeptidase-sensitive sequence.
  • the first cleavage domain and/or the second cleavage domain preferably contain a cleavage sequence of a protease or peptidase, since this can suppress non-specific cleavage.
  • the order of the first binding tag and the first cleavage domain can be set, for example, according to their positions relative to the first domain.
  • the first cleavage domain is positioned closer to the first domain than the first binding tag. This allows the first binding tag to be detached from the first domain when the first cleavage tag is cleaved in the production step described below. Therefore, in the production method of this embodiment, the first binding tag, the first cleavage domain, and the first domain are arranged in this order in the first protein, for example, from the N-terminus to the C-terminus.
  • the order of the first binding partner and the second cleavage domain can be set, for example, according to their positions relative to the second domain.
  • the second cleavage domain is positioned closer to the second domain than the first binding partner. This allows the first binding partner to be detached from the second domain when the second cleavage tag is cleaved in the production step described below. Therefore, in the production method of this embodiment, in the second protein, the first binding partner, the second cleavage domain, and the second domain are positioned in this order, for example, from the N-terminus to the C-terminus.
  • the first binding tag, the first cleavage domain, and the first domain are linked directly or indirectly to each other.
  • the first binding partner, the second cleavage domain, and the second domain are linked directly or indirectly to each other.
  • the direct bond means that the N- or C-terminal amino acid of a certain polypeptide or domain is bound to the C- or N-terminal amino acid of another polypeptide or domain by forming a peptide bond.
  • the indirect bond means that the N- or C-terminal amino acid of a certain polypeptide or domain is bound to the C- or N-terminal amino acid of another polypeptide or domain via a linker peptide (peptide linker), that is, the N- or C-terminal amino acid of a certain polypeptide or domain is bound to the C- or N-terminal amino acid of the linker peptide by forming a peptide bond, and the other end amino acid of the linker peptide is bound to the N- or C-terminal amino acid of the other polypeptide or domain.
  • a linker peptide peptide linker
  • the length of the linker peptide is, for example, 5 to 15 amino acids.
  • the linker peptide can be a known linker peptide, and specific examples include a GS linker (GS, GGS, or GGGGS (SEQ ID NO: 7)), a linker peptide having repeated GS linkers ([GS] l , [GGS] m , or [GGGGS] n (l, m, and n are each an integer of 2 or more)), GGGSGG (SEQ ID NO: 8), etc.
  • the first protein may include, for example, a soluble domain, a signal peptide, or other polypeptide on the N-terminus of the first binding tag.
  • the second protein may include, for example, a soluble domain, a signal peptide, or other polypeptide on the N-terminus of the first binding partner.
  • the soluble domain is preferably a polypeptide that, when fused with the polypeptide or protein, increases the expression level of the target protein, such as the first protein or the second protein, expressed in the transformant described below by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% or more, compared to when expressed as a polypeptide or protein not containing the soluble domain.
  • the soluble domain may be, for example, a protein or a partial polypeptide thereof. Specific examples of the soluble domain include GST, MBP, thioredoxin, antibodies, and antibody variants such as single-chain antibodies.
  • the number of solubility domains in the first protein may be one or more. In the latter case, the number of solubility domains may be one or more.
  • the number of solubility domains in the second protein may be one or more. In the latter case, the number of solubility domains may be one or more.
  • the first protein and the second protein may include, for example, a purification tag used for purifying the first protein, the second protein, or the heterodimeric protein.
  • the purification tag may be, for example, the affinity tag described above.
  • the purification tag may be added, for example, to at least one of the N-terminus (side) and C-terminus (side) of the first domain and the second domain.
  • the reaction conditions in the complex formation step may be any conditions under which the first protein and the second protein can form a dimer, and may be set appropriately taking into consideration the reaction conditions (binding conditions) of the binding tag and the binding partner, and/or the reaction conditions (binding conditions) of the first domain and the second domain.
  • the reaction temperature in the complex formation step is, for example, 4 to 50°C or 10 to 45°C.
  • the reaction time in the complex formation step is, for example, 0.1 to 24 hours or 0.5 to 12 hours.
  • the reaction pH in the complex formation step is, for example, pH 4 to 10 or pH 5 to 9.
  • the reaction conditions of the complex formation step can be set to reaction conditions under which a bond between the binding tag and the binding partner is sufficiently formed.
  • the complex formation step can be carried out, for example, at pH 5-8, at 4-37°C, in the presence of a buffer solution.
  • the complex formation step can be carried out, for example, at pH 5-8, at 4-37°C, in the presence of a buffer solution.
  • the reaction conditions of the complex formation step can be set, for example, based on the activity conditions of the enzyme used in the enzyme reaction.
  • the reaction conditions of the complex formation process can be set to reaction conditions under which a bond between the first domain and the second domain is sufficiently formed.
  • the reaction conditions of the complex formation process can be set to, for example, reaction conditions under which the disulfide bond is not reduced.
  • the reaction conditions of the complex formation process can be set to, for example, reaction conditions under which the isopeptide bond is not hydrolyzed.
  • reaction conditions in the complex formation step may further be conditions under which the first protein and the second protein can form disulfide bonds between the heavy chain and the light chain.
  • the manufacturing method of this embodiment may include a first purification step of purifying the first complex after the complex formation step.
  • the purification method in the purification step may be, for example, a general protein purification method such as chromatography.
  • the generation step the first cleavage domain and the second cleavage domain of the first complex are cleaved.
  • the bound first binding tag and first binding partner are released, and a heterodimer protein of the first domain and the second domain is generated.
  • the reaction conditions for the cleavage can be set to conditions under which the cleavage reaction occurs for the first cleavage domain and the second cleavage domain.
  • the reaction temperature for the cleavage is, for example, 0 to 40°C, 4 to 37°C, or 4 to 30°C.
  • the reaction time for the cleavage is, for example, 1 minute to 48 hours, 30 minutes to 48 hours, or 1 to 48 hours.
  • the reaction pH for the cleavage is, for example, pH 5 to 10, pH 6 to 9, or pH 6.5 to 9.
  • the generating step may be carried out in the presence of the protease or peptidase. In this case, the generating step can be carried out under reaction conditions under which the protease or peptidase exhibits cleavage activity.
  • the manufacturing method of the present disclosure may include a second purification step of purifying the heterodimer protein after the generation step.
  • the purification method in the purification step may be, for example, a general protein purification method such as chromatography.
  • the manufacturing method of this embodiment can produce a heterodimer protein from two proteins.
  • a first complex may be produced from two proteins in a host cell described below.
  • the production method of this embodiment can form the first complex in the host cell by expressing the first protein and the second protein in the host cell. Then, the production method of this embodiment can produce a heterodimeric protein, for example, by purifying the first complex from the host cell and carrying out the production step.
  • monomeric proteins are used as the two proteins, but the present disclosure is not limited to this, and one or both may be a protein of dimer or more, i.e., the multimeric protein.
  • the trimeric protein when producing a heterotrimeric protein as the heteromultimeric protein, the trimeric protein can be produced by making the first domain one protein of the trimeric proteins and the second domain a dimer of two proteins of the trimeric proteins. Therefore, any multimeric protein can be produced by the production method of the present disclosure.
  • the proteins for producing the heteromultimeric protein include a third protein and a fourth protein, and the heterotetrameric protein is produced using these. Therefore, unless otherwise specified, the description of the various configurations and steps in the production method of embodiment 2 can be used to refer to the description of the various configurations and steps in the production method of embodiment 1.
  • a third protein 103 and a fourth protein 104 are included.
  • a case where a heterotetramer protein 120 is manufactured using a first protein 101, a second protein 102, a third protein 103, and a fourth protein 104 is described as an example.
  • the first protein 101 includes a first binding tag 111, a first cleavage domain 112, and a first domain 113, in this order from the N-terminus to the C-terminus.
  • the second protein 102 includes a first binding partner 121 capable of binding to the first binding tag 111, a second cleavage domain 122, and a second domain 123, in this order from the N-terminus to the C-terminus.
  • the third protein 103 includes, in order from the N-terminus to the C-terminus, a second binding tag 131, a third cleavage domain 132, and a third domain 133.
  • the fourth protein 104 includes, in order from the N-terminus to the C-terminus, a second binding partner 141 capable of binding to the second binding tag 131, a fourth cleavage domain 142, and a fourth domain 143.
  • the first protein 101, the second protein 102, the third protein 103, and the fourth protein 104 are brought into contact with each other.
  • a bond is formed between the first domain 113 and the second domain 123, as shown by X1.
  • a bond is formed between the third domain 133 and the fourth domain 143, as shown by X2.
  • a bond is formed between the second domain 123 and the fourth domain 143, as shown by X3.
  • a bond is formed between the first binding tag 111 and the first binding partner 121, as shown by Y1, and a bond is formed between the second binding tag 131 and the second binding partner 141, as shown by Y2.
  • the first protein 101, the second protein 102, the third protein 103, and the fourth protein 104 form a second complex 110.
  • the first cleavage domain 112, the second cleavage domain 122, the third cleavage domain 132, and the fourth cleavage domain 142 of the second complex 110 are cleaved.
  • the first binding tag 111, the first binding partner 121, the second binding tag 131, and the second binding partner 141 which are arranged on the N-terminal side of the first cleavage domain 112, the second cleavage domain 122, the third cleavage domain 132, and the fourth cleavage domain 142, are detached from the second complex 110.
  • a heterotetrameric protein 120 including the first domain 113, the second domain 123, the third domain 133, and the fourth domain 143 can be produced. Therefore, according to the manufacturing method of this embodiment, it is presumed that the desired heterotetramer protein can be produced by arranging the monomer proteins that constitute the desired heterotetramer in the first domain 113, the second domain 123, the third domain 133, and the fourth domain 143.
  • the second complex is formed by binding the first binding tag to the first binding partner, binding the second binding tag to the second binding partner, binding the first domain to the second domain, binding the second domain to the fourth domain, and binding the third domain to the fourth domain.
  • the first protein, the second protein, the third protein, and the fourth protein can be prepared by genetic engineering techniques, for example, as described in the protein manufacturing method of the present disclosure below.
  • the manufacturing method of the present disclosure may optionally include a first expression step of expressing the first protein, the second protein, the third protein, and the fourth protein in a host cell prior to the complex formation step.
  • the expression method in the expression step can be based on the description of the protein, nucleic acid, expression vector, transformant, and protein manufacturing method of the present disclosure below.
  • the first protein, the second protein, the third protein, and the fourth protein are reacted.
  • the first protein, the second protein, the third protein, and the fourth protein form a second complex
  • the first binding tag and the first binding partner bind
  • the second binding tag and the second binding partner bind
  • the first domain binds to the second domain
  • the second domain binds to the fourth domain
  • the third domain binds to the fourth domain, thereby forming the second complex.
  • the complexation of the first to fourth proteins may be, for example, (1) caused by binding between the binding tag and the binding partner, i.e., binding between the first binding tag and the first binding partner and/or binding between the second binding tag and the second binding partner, or (2) caused by binding between the domains, i.e., binding between the first domain and the second domain, binding between the second binding domain and the fourth domain, and/or binding between the third domain and the fourth domain, or may be caused by binding between both (1) and (2).
  • the complexation is caused by binding between both (1) and (2) because this can improve the dimer formation ability.
  • the bond between the first domain and the second domain, the bond between the second binding domain and the fourth domain, and/or the bond between the third domain and the fourth domain may be a direct bond, an indirect bond (association), or may be formed by both a direct bond and an indirect bond between the first domain and the second domain, but is preferably a direct bond.
  • the direct bond is a covalent bond, and specific examples thereof include an amide bond (peptide bond, isopeptide bond, etc.) between amino acids and a disulfide bond between cysteines.
  • the indirect bond is a non-covalent bond, and specific examples thereof include a hydrogen bond, a hydrophobic bond, etc.
  • the first to fourth domains can adopt amino acid sequences capable of forming a tetramer, condition-dependent or condition-independent, when proteins including each domain coexist.
  • the first to fourth domains can utilize amino acid sequences of motif sequences forming each subunit or dimer in a protein tetramer.
  • the first to fourth domains can utilize amino acid sequences of motif sequences forming each subunit or tetramer in a protein multimer.
  • the protein tetramer can be a heterotetramer containing some of the same subunits, or a heterotetramer in which all subunits are different.
  • the protein tetramer examples include immunoglobulins (antibodies) such as IgA, IgD, IgE, IgG, and IgM; small bispecific antibodies; and the like.
  • antibody subunits are used as the first to fourth domains, the antibody may be an antibody with a modified constant region as described above.
  • the first domain is a light chain of an antibody that binds to a first target
  • the second domain is a heavy chain of an antibody that binds to the first target
  • the third domain is a light chain of an antibody that binds to a second target
  • the fourth domain is a heavy chain of an antibody that binds to the second target.
  • the antibody that binds to the first target and the antibody that binds to the second target may recognize the same antigen or different antigens, but it is preferable that they recognize different antigens.
  • the antibody that binds to the first target and the antibody that binds to the second target may recognize the same epitope or different epitopes, but it is preferable that they recognize different epitopes.
  • the manufacturing method of the present disclosure can suitably manufacture, for example, bispecific antibodies.
  • the first to fourth domains use the motif sequence for tetramer formation of the antibody, and the first and third domains are antibody light chains, and the second and fourth domains are antibody heavy chains, it is preferable that the second and fourth domains use antibody heavy chains in which the constant region that specifically associates with the antibody has been modified. Examples of the antibody in which the constant region that specifically associates with the antibody has been modified include charge pair and knobs-in-holes.
  • the first binding tag and the first binding partner are molecules that, when a protein containing the first binding tag and a protein containing the first binding partner coexist, bind to the first binding tag condition-dependently or condition-independently.
  • the binding between the first binding tag and the first binding partner may be direct or indirect.
  • the second binding tag and the second binding partner are molecules that, when a protein containing the second binding tag and a protein containing the second binding partner coexist, bind to the first binding tag condition-dependently or condition-independently.
  • the binding between the second binding tag and the second binding partner may be direct or indirect.
  • the binding between the first binding tag and the first binding partner and the binding between the second binding tag and the second binding partner are preferably direct. As a result, the manufacturing method of this embodiment can suppress, for example, nonspecific binding between the binding tag and the binding partner, allowing the desired heterotetramer to be produced efficiently.
  • Specific examples of the second binding tag and the second binding partner can be the same as the examples of the first binding tag and the first binding partner in embodiment 1.
  • the first binding tag and the second binding tag are configured to be capable of binding to, for example, the first binding partner and the second binding partner, respectively. That is, the binding between the first binding tag and the second binding partner is configured to be less specific than the binding between the first binding tag and the first binding partner, and the binding between the second binding tag and the first binding partner is configured to be less specific than the binding between the second binding tag and the second binding partner.
  • the first binding tag and the first binding partner, and the second binding tag and the second binding partner are different combinations of binding tags and binding partners.
  • the second binding tag may be one or more. In the latter case, the second binding tag may be one type or more types. In the third protein, the second binding tag is preferably located on the N-terminus of the third protein.
  • the second binding partner may be one or more. In the latter case, the second binding partner may be one type or more types. In the fourth protein, the second binding partner is preferably located on the N-terminus side of the fourth protein.
  • the number of second binding tags in the third protein and the number of second binding partners in the fourth protein may be the same or different, but are preferably the same.
  • the second binding tag and the second binding partner are interchangeable, and the combinations described above may be used interchangeably.
  • the first to fourth cleavage domains are domains in which cleavage occurs condition-dependently or condition-independently.
  • the cleavage is carried out in the production step described below after the formation of the second complex. For this reason, it is preferable that the first to fourth cleavage domains are cleavage domains in which cleavage occurs condition-dependently.
  • the third cleavage domain and the fourth cleavage domain can be derived from the explanation of the first cleavage domain and the second cleavage domain in embodiment 1.
  • the first to fourth cleavage domains preferably contain the cleavage sequence of the protease or peptidase, since they can suppress non-specific cleavage.
  • the first to fourth cleavage domains may be partly or entirely the same cleavage domain, or partly or entirely different cleavage domains. Furthermore, in the first to fourth proteins, the first to fourth cleavage domains may each be one or more. In the latter case, the cleavage domain may be one type or multiple types. By using the same cleavage domain for the first to fourth cleavage domains, for example, in the production process described below, the first to fourth cleavage domains can be cleaved in a single reaction, and therefore a tetramer-dimer protein can be produced efficiently.
  • the order of the second binding tag and the third cleavage domain can be set, for example, according to their positions relative to the third domain.
  • the third cleavage domain is positioned closer to the third domain than the second binding tag. This allows the second binding tag to be detached from the third domain when the third cleavage tag is cleaved in the production step described below. Therefore, in the production method of this embodiment, in the third protein, the second binding tag, the third cleavage domain, and the third domain are positioned in this order, for example, from the N-terminus to the C-terminus.
  • the order of the second binding partner and the fourth cleavage domain can be set, for example, according to their positions relative to the fourth domain.
  • the fourth cleavage domain is positioned closer to the fourth domain than the second binding partner. This allows the second binding partner to be detached from the fourth domain when the fourth cleavage tag is cleaved in the production step described below. Therefore, in the production method of this embodiment, in the fourth protein, the second binding partner, the fourth cleavage domain, and the fourth domain are arranged in this order, for example, from the N-terminus to the C-terminus.
  • the second binding tag, the third cleavage domain, and the third domain are each directly or indirectly linked.
  • the second binding partner, the fourth cleavage domain, and the fourth domain are each directly or indirectly linked.
  • the direct and indirect bonds can be explained by referring to the explanation of the direct and indirect bonds in the explanation of the first and second proteins in embodiment 1.
  • the third protein may, for example, include other polypeptides, such as the solubility domain and the signal peptide, on the N-terminal side of the second binding tag.
  • the fourth protein may, for example, include other polypeptides, such as the solubility domain and the signal peptide, on the N-terminal side of the second binding partner.
  • the number of solubility domains in the third protein may be one or more. In the latter case, the number of solubility domains may be one or more.
  • the number of solubility domains in the fourth protein may be one or more. In the latter case, the number of solubility domains may be one or more.
  • the third protein and the fourth protein may include, for example, a purification tag used for purifying the third protein, the fourth protein, or the heterotetrameric protein.
  • the purification tag may be, for example, the affinity tag described above.
  • the purification tag may be added, for example, to at least one of the N-terminus (side) and C-terminus (side) of the third domain and the fourth domain.
  • the reaction conditions in the complex formation step may be any conditions under which the first to fourth proteins can form a tetramer, and can be set appropriately taking into consideration the reaction conditions (binding conditions) of the binding tag and the binding partner, and/or the reaction conditions (binding conditions) of the first to fourth domains.
  • the reaction conditions of the complex formation process can be set to reaction conditions under which the bond between the third domain and the fourth domain is sufficiently formed.
  • the reaction conditions of the complex formation process can be set to, for example, reaction conditions under which the disulfide bond is not reduced.
  • the reaction conditions of the complex formation process can be set to, for example, reaction conditions under which the isopeptide bond is not hydrolyzed.
  • the reaction conditions of the complex formation process can be set to reaction conditions under which the bond between the second domain and the fourth domain is sufficiently formed.
  • the reaction conditions of the complex formation process can be set to, for example, reaction conditions under which the disulfide bond is not reduced.
  • the reaction conditions in the complex formation step can be set such that the isopeptide bond is not hydrolyzed.
  • the reaction conditions in the complex formation step may further be conditions under which the first to fourth proteins can form disulfide bonds between the heavy chains and the light chains, and between the heavy chains.
  • the manufacturing method of the present disclosure may include a first purification step of purifying the second complex after the complex formation step.
  • the purification method in the purification step may be, for example, a general protein purification method such as chromatography.
  • the first cleavage domain, the second cleavage domain, the third cleavage domain, and the fourth cleavage domain of the second complex are cleaved.
  • the bound first binding tag and first binding partner, and the bound second binding tag and second binding partner are detached, and a heterotetramer of the first domain, the second domain, the third domain, and the fourth domain is generated.
  • reaction conditions for the cleavage e.g., reaction temperature, reaction time, reaction pH, etc.
  • reaction temperature, reaction time, reaction pH, etc. can be set to conditions under which the cleavage reaction occurs for the first to fourth cleavage domains.
  • the generating step may be carried out in the presence of the protease or peptidase. In this case, the generating step can be carried out under reaction conditions under which the protease or peptidase exhibits cleavage activity.
  • the manufacturing method of the present disclosure may include a second purification step of purifying the heterotetrameric protein after the generation step.
  • the purification method in the purification step may be, for example, a general protein purification method such as chromatography.
  • the manufacturing method of this embodiment can produce a heterotetrameric protein from four proteins.
  • the production method of this embodiment an example of producing a heterodimeric protein using four isolated proteins has been described, but the present disclosure is not limited to this, and a second complex may be produced from four proteins in a host cell described below.
  • the production method of this embodiment can form the second complex in the host cell by expressing the first to fourth proteins in the host cell. Then, the production method of this embodiment can produce a heterotetrameric protein, for example, by purifying the second complex from the host cell and carrying out the production step.
  • each of the four proteins is a monomeric protein, but the present disclosure is not limited to this, and any one or both may be a dimeric or higher protein, i.e., the multimeric protein.
  • the second protein and the fourth protein associate and bind only through the binding of the second domain and the fourth domain, but the second protein and the fourth protein may be configured to be able to bind specifically via other domains.
  • the second protein may further include a fifth cleavage domain and a third binding tag, in this order, at the C-terminus.
  • the fourth protein may further include a sixth cleavage domain and a third binding partner capable of binding to the third binding tag, in this order, at the C-terminus.
  • the third binding tag and the third binding partner further bind.
  • the generation step for example, the fifth cleavage domain and the sixth cleavage domain in the second complex are further cleaved.
  • the first protein 201, the second protein 202, the third protein 203, and the fourth protein 204 are brought into contact with each other.
  • This causes binding between the binding tags and binding partners of each protein.
  • the first binding tag 211 and the first binding partner 221 bind.
  • the second binding tag 231 and the second binding partner 241 bind.
  • the third binding tag 251 and the third binding partner 261 bind.
  • binding also occurs between the first domain 213, the second domain 223, the third domain 233, and the fourth domain 243 of each protein.
  • the first domain 213 and the second domain 223 bind. Additionally, as indicated by X2, the third domain 233 binds to the fourth domain 243. Furthermore, as indicated by X3, the second domain 223 binds to the fourth domain 243. As a result, in the complex formation step, the first protein 201, the second protein 202, the third protein 203, and the fourth protein 204 form the second complex 210.
  • the first cleavage domain, the second cleavage domain, the third cleavage domain, the fourth cleavage domain, the fifth cleavage domain, and the sixth cleavage domain of the second complex are cleaved.
  • the bound first binding tag 211 and first binding partner 221, the bound second binding tag 231 and second binding partner 241, and the bound third binding tag 251 and third binding partner 261 are detached, and a heterotetramer 220 of the first domain 213, the second domain 223, the third domain 233, and the fourth domain 243 is generated.
  • the third binding tag and the third binding partner can be the same as those of the first binding tag and the first binding partner in embodiment 1.
  • the first binding tag, the second binding tag, and the third binding tag are configured to be capable of binding to the first binding partner, the second binding partner, and the third binding partner, respectively, for example.
  • the first binding tag and the first binding partner, the second binding tag and the second binding partner, and the third binding tag and the third binding partner are different combinations of binding tags and binding partners.
  • the fifth cleavage domain and the sixth cleavage domain can be the same as the examples of the first cleavage domain and the sixth cleavage domain in embodiment 1. It is preferable that the first to sixth cleavage domains are the same cleavage domain.
  • the present disclosure provides a protein that can be suitably used for producing the heteromultimeric protein.
  • the protein of the present disclosure is the first protein, the second protein, the third protein, and/or the fourth protein described below.
  • the protein of the present disclosure (first protein) comprises, from the N-terminus to the C-terminus, a first binding tag capable of binding to a first binding partner, a first cleavage domain, and a first domain, in that order.
  • the protein of the present disclosure (the second protein) comprises, from the N-terminus to the C-terminus, a first binding partner capable of binding to a first binding tag, a second cleavage domain, and a second domain, in that order.
  • the protein of the present disclosure includes, from the N-terminus to the C-terminus, a second binding tag capable of binding to a second binding partner, a third cleavage domain, and a third domain, in that order.
  • the protein of the present disclosure (fourth protein) comprises, from the N-terminus to the C-terminus, a first binding partner capable of binding to a second binding tag, a fourth cleavage domain, and a fourth domain, in that order.
  • the second protein and/or the fourth protein of the present disclosure may further comprise, at the C-terminus, a cleavage domain and a binding tag or binding partner, in that order.
  • the first domain of the first protein and/or the third domain of the third protein of the present disclosure are, for example, each a polypeptide including an amino acid sequence of an immunoglobulin light chain region.
  • the second domain of the second protein and/or the fourth domain of the fourth protein are, for example, each a polypeptide including an amino acid sequence of an immunoglobulin heavy chain region.
  • the present disclosure provides a heteromultimeric protein that can be suitably used for producing the heteromultimeric protein.
  • the protein (heteromultimeric protein) of the present disclosure includes two proteins, The two proteins include a first protein and a second protein, the first protein comprises, in order from N-terminus to C-terminus, a first binding tag, a first cleavage domain, and a first domain; the second protein comprises, in order from N-terminus to C-terminus, a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain; the first protein and the second protein form a dimer by binding between the first domain and the second domain; The first binding tag and the first binding partner are bound to each other.
  • the heteromultimeric protein of the present disclosure further comprises a third protein and a fourth protein, the third protein comprises, in order from N-terminus to C-terminus, a second binding tag, a third cleavage domain, and a third domain; the fourth protein comprises, in order from N-terminus to C-terminus, a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain; the second protein and the fourth protein form a dimer by binding between the second domain and the fourth domain; the third protein and the fourth protein form a dimer by binding between the third domain and the fourth domain; the first binding tag and the first binding partner bind; Preferably, said second binding tag and said second binding partner are linked.
  • the first domain of the first protein and the third domain of the third protein are each polypeptides that include an amino acid sequence of an immunoglobulin light chain.
  • the second domain of the second protein and the fourth domain of the fourth protein are each polypeptides that include an amino acid sequence of an immunoglobulin heavy chain.
  • nucleic acids that can be used to synthesize heteromultimeric proteins.
  • the nucleic acids of the present disclosure encode the proteins and/or heteromultimeric proteins of the present disclosure.
  • the nucleic acid of the present disclosure may, for example, encode one or more of the proteins and heteromultimeric proteins of the present disclosure, or may encode more than one.
  • the nucleic acid of the present disclosure can be designed by substituting the corresponding codons based on the amino acid sequence of the heteromultimeric protein of the present disclosure.
  • the base sequence of the nucleic acid of the present disclosure may be, for example, codon-optimized, and is preferably codon-optimized for the host cell described below.
  • the present disclosure provides an expression vector that can be used to synthesize a protein and/or a heteromultimeric protein.
  • the expression vector of the present disclosure contains the nucleic acid of the present disclosure.
  • the protein and/or the heteromultimeric protein of the present disclosure (hereinafter, also referred to as the "material protein of the present disclosure") can be suitably produced by genetic engineering techniques.
  • the expression vector of the present disclosure may contain, for example, a nucleic acid encoding one or more of the proteins and heteromultimeric proteins of the present disclosure, and may contain a nucleic acid encoding more than one of them.
  • the expression vector of the present disclosure is, for example, an expression vector into which the nucleic acid of the present disclosure is inserted.
  • the expression vector refers to, for example, a nucleic acid molecule that can transport an inserted gene into a target such as a cell.
  • the expression vector is not particularly limited in its configuration, so long as it contains a polynucleotide encoding the material protein of the present disclosure so that the material protein of the present disclosure encoded by the polynucleotide of the nucleic acid of the present disclosure can be expressed.
  • the material protein of the present disclosure may be inserted, for example, in part or in whole, into the same expression vector, or into separate expression vectors.
  • the expression vectors of the present disclosure may be configured as an expression vector set including an expression vector containing a nucleic acid encoding the material protein of the present disclosure.
  • the expression vector can be prepared, for example, by inserting a polynucleotide encoding the material protein of the present disclosure, i.e., the nucleic acid of the present disclosure, into a backbone vector (hereinafter also referred to as a "basic vector").
  • a backbone vector hereinafter also referred to as a "basic vector”.
  • the type of the expression vector is not particularly limited and can be appropriately determined depending on, for example, the type of the host.
  • Examples of the host cell include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells.
  • Examples of the prokaryotic organisms include bacteria such as Escherichia genus such as Escherichia coli , and Pseudomonas genus such as Pseudomonas putida .
  • Examples of the eukaryotic organisms include yeasts such as Saccharomyces cerevisiae .
  • Examples of the animal cells include HEK293 cells, Expi293F cells, COS cells, CHO cells, and the like, and examples of the insect cells include Sf9 and Sf21.
  • the expression vector may be a viral vector or a non-viral vector.
  • the expression vector may be, for example, a binary vector.
  • the expression vector may be, for example, pETDuet-1, pQE-80L, pUCP26Km, etc.
  • the expression vector may be, for example, pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8, etc.
  • the expression vector may be, for example, pYepSec1, pMFa, pYES2, etc.
  • the expression vector When transforming insect cells, the expression vector may be, for example, pAc, pVL, etc.
  • examples of the expression vector include pcDNA3.1, pcDNA3.4, pCAG, pCAGEN, pCDM8, and pMT2PC.
  • the expression vector preferably has a regulatory sequence that regulates, for example, the expression of the polynucleotide encoding the material protein of the present disclosure and the expression of the material protein of the present disclosure encoded by the polynucleotide of the material protein of the present disclosure.
  • the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori).
  • the arrangement of the regulatory sequence in the expression vector is not particularly limited.
  • the regulatory sequence may be arranged in a manner that allows functional regulation of the expression of the polynucleotide encoding the material protein of the present disclosure and the expression of the material protein of the present disclosure encoded by the polynucleotide, and may be arranged based on a known method.
  • the regulatory sequence may utilize a sequence that is already included in the basic vector, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence included in the basic vector may be replaced with another regulatory sequence.
  • the expression vector may further include, for example, a coding sequence for a selection marker.
  • a selection marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker.
  • the insertion of DNA, the insertion of the regulatory sequence, and/or the insertion of the coding sequence of the selection marker into the expression vector may be carried out, for example, by a method using restriction enzymes and ligase, or by using a commercially available kit, etc.
  • transformant of the present disclosure contains a nucleic acid encoding the material protein of the present disclosure.
  • the transformant of the present disclosure can suitably produce the material protein of the present disclosure.
  • the method for producing a transformant disclosed herein also includes a step of introducing the nucleic acid disclosed herein into a host. According to the method for producing a transformant disclosed herein, the transformant can be produced.
  • the explanation of the nucleic acid encoding the material protein of the present disclosure can be applied to the nucleic acid encoding the material protein of the present disclosure.
  • the expression vector of the present disclosure may be used as the nucleic acid of the present disclosure.
  • the nucleic acid of the present disclosure exists as an exogenous molecule. Therefore, the transformant of the present disclosure can be produced, for example, by introducing the nucleic acid of the present disclosure into the host.
  • the method of introducing the nucleic acid is not particularly limited and can be performed by a known method.
  • the nucleic acid may be introduced, for example, by the expression vector.
  • the introduction method can be appropriately set, for example, depending on the type of the host. Examples of the introduction method include an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method via Agrobacterium, and the like.
  • a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-
  • liposome examples include lipofectamine and cationic liposome
  • introduction aid examples include atelocollagen, nanoparticles, and polymers.
  • the host is a microorganism, a method via E. coli or Ps. putida is preferable.
  • the polynucleotide encoding the protein of the present invention may be introduced into the host, for example, by the expression vector of the present disclosure.
  • the present disclosure provides a method for producing a protein and/or a heteromultimeric protein that can be suitably used for producing a protein and/or a heteromultimeric protein.
  • the method for producing a protein of the present disclosure includes an expression step of expressing the nucleic acid of the present disclosure, the expression vector of the present disclosure, and/or the expression vector set of the present disclosure.
  • the material protein of the present disclosure can be produced.
  • the material protein of the present disclosure may be expressed, for example, using the expression vector of the present disclosure.
  • the method for expressing the material protein of the present disclosure is not particularly limited, and any known method can be used.
  • a host may be used, or a cell-free protein synthesis system may be used.
  • the host into which the material protein of the present disclosure or a nucleic acid encoding the same has been introduced it is preferable to use the host into which the material protein of the present disclosure or a nucleic acid encoding the same has been introduced, and to express the material protein of the present disclosure in the host by culturing the host.
  • a nucleic acid encoding the material protein of the present disclosure into a host, a transformant that synthesizes the material protein of the present disclosure can be produced, and the material protein of the present disclosure can be synthesized by culturing the transformant.
  • the method for culturing the host is not particularly limited and can be set appropriately depending on the type of the host.
  • the medium used for culturing is not particularly limited and can be determined appropriately depending on the type of the host.
  • the polynucleotide of the material protein of the present disclosure in a cell-free protein synthesis system.
  • an expression vector may be used to express the polynucleotide of the material protein of the present disclosure.
  • the cell-free protein synthesis system can be carried out by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which a polynucleotide encoding the material protein of the present disclosure has been introduced, and for example, a commercially available reagent kit can be used.
  • the method for producing a protein of the present disclosure may include, for example, a recovery step of recovering the material protein of the present disclosure.
  • the material protein of the present disclosure obtained in the recovery step may be, for example, a crude product or a purified protein.
  • the recovery step involves, for example, removing insoluble matter by filtering or centrifuging the culture supernatant. Then, in the recovery step, the culture supernatant after removal of the insoluble matter can be separated and purified using an appropriate combination of concentration using an ultrafiltration membrane; salting out using ammonium sulfate precipitation or the like; dialysis; and chromatography using various columns such as an ion exchange column and a gel filtration column to obtain the material protein of the present disclosure.
  • the recovery step involves, for example, disrupting the transformant by pressure treatment, ultrasonic treatment, or the like.
  • the resulting disruption solution is then subjected to removal of insoluble matter, separation, and purification as described above to obtain the material protein of the present disclosure.
  • the material protein of the present disclosure obtained by the manufacturing method of the present disclosure may be used, for example, as a crudely purified protein as it is, or as a partially purified protein, or as a single purified protein.
  • the manufacturing method of the present disclosure may powder the obtained material protein of the present disclosure, for example, by freeze-drying, vacuum drying, or spray drying.
  • the manufacturing method of the present disclosure may dissolve the protein of the present invention in advance in a buffer solution such as acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD's buffer (e.g., HEPES, PIPES, MES, MOPS, etc.).
  • a plasmid vector capable of expressing the following four proteins was constructed.
  • First protein a protein comprising a soluble domain, a SpyTag (binding tag), and a light chain of a CD3 antibody (VHH-SpyTag-CD3 L chain)
  • Second protein a protein containing a soluble domain, SpyCatcher (binding partner), and the heavy and variable domains of the CD3 antibody (VHH-SpyCatcher-CD3 H chain)
  • the third protein is the light chain of the Herceptin antibody.
  • the fourth protein is the heavy chain of the Herceptin antibody.
  • a plasmid vector capable of expressing VHH-SpyTag-CD3 L chain was constructed by the following procedure.
  • a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide, aGFP4 (single-chain antibody (soluble domain), SEQ ID NO: 9), G1 linker, SpyTag (SEQ ID NO: 2), thrombin cleavage sequence, M291 light chain variable region, and M291 light chain constant region was amplified by PCR.
  • the resulting full length synthetic gene was linked to an expression vector (pCDNA3.4) for animal cells to construct an expression vector for the recombinant protein.
  • the expression vector contains the VHH-SpyTag-CD3 L chain region (SEQ ID NO: 10) in the following order from the N-terminus to the C-terminus: signal peptide, aGFP4, G1 linker, SpyTag, thrombin cleavage sequence, M291 light chain variable region, and M291 light chain constant region, as shown in brackets.
  • aGFP4 (SEQ ID NO: 9) QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS
  • VHH-SpyTag-CD3 L chain region (SEQ ID NO: 10) [MEFGLSWLFLVAILKGVQC][QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS][GGSGG][AHIVMVDAYKPTK][GGSGGGGSGG][LVPRGSHMHM][DIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYKQKSGTSPKRWTYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGSGTKLEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
  • a plasmid vector capable of expressing VHH-SpyCatcher-CD3 H chain was constructed by the following procedure. First, the full length of a synthetic gene (Eurofins Genomics) containing base sequences encoding the signal peptide, Ia1 (single-chain antibody (soluble domain), sequence number 11), G1 linker, SpyCatcher (sequence number 1), G2 linker, thrombin cleavage sequence, M291 heavy chain variable region, M291 heavy chain constant region, hinge region, and Fc region was amplified by PCR. The resulting full length synthetic gene was linked to an animal cell expression vector (pCDNA3.4) to construct an expression vector for the recombinant protein.
  • pCDNA3.4 animal cell expression vector
  • the expression vector is a VHH-SpyCatcher-CD3 H chain region (SEQ ID NO: 12) in which, from the N-terminus to the C-terminus, as shown in parentheses, a signal peptide, Ia1, G1 linker, SpyCatcher, G2 linker, thrombin cleavage sequence, M291 heavy chain variable region, M291 heavy chain constant region, hinge region, and Fc region are linked in this order.
  • Ia1 (SEQ ID NO: 11) QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS
  • VHH-SpyCatcher-CD3 H chain region (SEQ ID NO: 12) [MEFGLSWLFLVAILKGVQC][QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GGSGG][DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG][GGSGGGGSGG][LVPRGSHMHM][QVQLQQSGAELARPGASVKMSCKASGYTFISYTMHWVKQRPGQGLEWIGYINPRSGYTHYNQKLKDKATLTADKSSSSAYMQLSSLTSEDYAVYY CARSAYYDYDGFAY
  • a plasmid vector capable of expressing the light chain of the Herceptin antibody was constructed by the following procedure. First, the full length of a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide, the light chain variable region of h4D5, and the light chain constant region of h4D5 was amplified by PCR. The resulting full length synthetic gene was linked to an expression vector (pCAGGS) for animal cells to construct an expression vector for the recombinant protein.
  • pCAGGS expression vector for animal cells
  • the signal peptide, the light chain variable region of h4D5, and the light chain constant region of h4D5 are linked in this order from the N-terminus to the C-terminus as the light chain region of the Herceptin antibody (SEQ ID NO: 13), as shown in parentheses.
  • Herceptin antibody light chain region (SEQ ID NO: 13) [METPAQLLFLLLWLPESTG][DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]
  • a plasmid vector capable of expressing the heavy chain of the Herceptin antibody was constructed as follows. First, a synthetic gene (Eurofin Genomics) containing a base sequence encoding a signal peptide, a heavy chain variable region of h4D5, a heavy chain constant region of h4D5, a hinge region, and an Fc region of h4D5 was amplified in its entirety by PCR. The resulting full-length synthetic gene was linked to an expression vector (pCAGGS) for animal cells to construct an expression vector for a recombinant protein.
  • pCAGGS expression vector for animal cells to construct an expression vector for a recombinant protein.
  • the expression vector contains the signal peptide, the heavy chain variable region of h4D5, the heavy chain constant region of h4D5, a hinge region, and an Fc region of h4D5 linked in this order from the N-terminus to the C-terminus as the heavy chain region of the Herceptin antibody (SEQ ID NO: 14), as shown in brackets.
  • Herceptin antibody heavy chain region (SEQ ID NO: 14) [MEFGLSWLFLVAILKGVQC][EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KV][EPKSCDKTH][TCPPCP][APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKALGQPREPQVYTLPPCRDELTKNQVSLWCLVK
  • the four plasmid expression vectors of the recombinant proteins were transfected into Expi293F cells.
  • the frozen Expi293F cells were thawed and seeded in HE400 medium. After the seeding, the cells were shake-cultured under conditions of 37°C, 8% CO 2 , and 125 rpm until the cell number reached 3-5 ⁇ 10 6 /ml and the survival rate reached 95% or more. After the culture, the cells were added to 25 ml of HE400 medium so that the cell number reached 75 ⁇ 10 6 cells. After the addition, the total amount of the plasmid vector was added to Opti-MEM (registered trademark) to a final concentration of 30,000 ng/ml.
  • Opti-MEM registered trademark
  • the collected elution fraction was placed in a dialysis membrane and dialyzed with a dialysis buffer (150 mmol/l NaCl, 50 mmol/l HEPES). After the dialysis, the column was concentrated using a concentration tube to obtain a purified sample.
  • a dialysis buffer 150 mmol/l NaCl, 50 mmol/l HEPES.
  • Figure 4 is a photograph showing the results of SDS-PAGE.
  • the left side of the photograph shows molecular weight (kDa).
  • a band corresponding to the estimated molecular weight of the complex was detected in the purified sample.
  • Bands corresponding to the estimated molecular weights of the Herceptin antibody light chain and the Herceptin antibody heavy chain were also detected.
  • Example 1 (3) was a complex containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain.
  • protein purification was performed in the same manner as in Example 1 (2), and the purified eluate fraction was added to a thrombin buffer (2.5 mmol/l CaCl 2 , 150 mmol/l NaCl, 20 mmol/l Tris-HCl (pH 8.0)). After the addition, dialysis was performed for 6 hours.
  • a thrombin buffer 2.5 mmol/l CaCl 2 , 150 mmol/l NaCl, 20 mmol/l Tris-HCl (pH 8.0)
  • thrombin (cleavage ability 2 units/ ⁇ l, manufactured by Wako Pure Chemical Industries, Ltd.) was added according to the yield of the protein. After the addition, the mixture was left to stand at 25° C. overnight. After the standing, the sample after the cleavage reaction was purified using a protein A column. After the purification, a thrombin-cleaved purified sample was obtained. Then, SDS-PAGE was performed. The SDS-PAGE was performed in the same manner as in Example 1(3) above, except that the purified thrombin-cleaved purified sample was used in addition to the purified sample. The results are shown in FIG.
  • Figure 5 is a photograph showing the results of SDS-PAGE.
  • the top of the photograph shows the type of sample, and the left side of the photograph shows the molecular weight (kDa).
  • the molecular weight (kDa).
  • no bands corresponding to the estimated molecular weight of the complex were detected in the thrombin-cleaved purified sample.
  • bands corresponding to the estimated molecular weights of the Herceptin antibody light chain, the Herceptin antibody heavy chain, the CD3 antibody light chain, the CD3 antibody heavy chain, and the bound VHH-SpyTag and VHH-SpyCatcher were detected in the thrombin-cleaved purified sample.
  • Herceptin was separated from the purified sample obtained in Example 1 (3) by size exclusion chromatography. Specifically, the protein purified in Example 1 (3) was passed through a gel filtration chromatography column (Superdex 200 increase 30/100 GL, manufactured by GE Healthcare) at 0.5 mL/min, and the absorbance at 212 nm was measured at room temperature (about 25° C.). In the measurement, 1 ⁇ PBS was used as a buffer. The results are shown in FIG. 6.
  • Figure 6 is a graph showing the elution pattern of Herceptin by size exclusion chromatography.
  • the horizontal axis indicates the exclusion time (hours) and the vertical axis indicates the absorbance.
  • elution of Herceptin obtained by the manufacturing method disclosed herein was confirmed (arrow in Figure 6).
  • Example 2 After the centrifugation, the supernatant was removed by aspirating, and 1x PBS and the CD3 antibody purified in the same manner as in Example 1 (5) were added to one microtube (b) so that the final concentration of the CD3 antibody was 0.05 ⁇ mol/l, and the mixture was mixed by inversion. After the mixture was mixed by inversion, the mixture was left to stand for 20 minutes. After the standing, the mixture was centrifuged at 2000 rpm, 25°C, and 7 minutes.
  • the mixture was centrifuged again under the conditions of 2000 rpm, 25° C., and 7 minutes. After the centrifugation, the supernatant was aspirated and removed by aspirator, and 1 ml of 1 ⁇ PBS was added and suspended. After the suspension, sterilization was performed using a mesh filter. After setting the measurement conditions, the negative control (c), the positive control (a), and the sample (b) were measured in this order using a cell analyzer RF-500 (Sysmex Corporation). After the measurements, the measurement results were graphed using an FCSalyzer. These results are shown in FIG.
  • FIG. 7 is a graph showing the binding of the CD3 antibody light chain and the CD3 antibody heavy chain to CD3-positive cells by flow cytometry.
  • the horizontal axis shows the fluorescence intensity
  • the vertical axis shows the cell count.
  • two microtubes (d) and (e) were centrifuged under conditions of 2000 rpm, 25°C, and 7 minutes. After the centrifugation, the supernatant was aspirated and removed using an aspirator, and 1 ml of 1x PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again under the conditions of 2000 rpm, 25° C., and 7 minutes. After the centrifugation, the supernatant was removed by aspirating with an aspirator.
  • the supernatant was removed by aspirating, and 1 ml of 1 ⁇ PBS was added.
  • the two microtubes (d) and (e) were centrifuged again under the conditions of 2000 rpm, 25° C., and 7 minutes.
  • the supernatant was removed by aspirating, and 1 ⁇ l of anti-Fc-FITC (manufactured by AbCam) and 1 ml of 1 ⁇ PBS were added to the two microtubes (d) and (e), and mixed by inversion. After the inversion, the mixture was left to stand for 20 minutes.
  • the two microtubes were centrifuged under the conditions of 2000 rpm, 25° C., and 7 minutes. After the centrifugation, the supernatant was removed by aspirating, and 1 ml of 1 ⁇ PBS was added. After the addition, the two microtubes (d) and (e) were centrifuged again under the conditions of 2000 rpm, 25° C., and 7 minutes. After the centrifugation, the supernatant was removed by suction using an aspirator, and 1 ml of 1 ⁇ PBS was added and suspended. After the suspension, sterilization was performed using a mesh filter.
  • FIG. 8 is a graph showing the binding of the Herceptin antibody light chain and the Herceptin antibody heavy chain to HER2-positive cells by flow cytometry.
  • the horizontal axis shows the fluorescence intensity
  • the vertical axis shows the cell count.
  • Herceptin antibody light chain and the Herceptin antibody heavy chain obtained by the method of the present disclosure bind to Herceptin antibody light chain and Herceptin antibody heavy chain obtained by the method of the present disclosure bind to Her2-positive breast cancer cells.
  • First protein a protein comprising a variable domain, a SpyTag (binding tag), and a light chain of the CD3 antibody (VHH-SpyTag-CD3 L chain)
  • Second protein a protein comprising SpyCatcher (binding partner) and the heavy and variable domains of the CD3 antibody (VHH-SpyCatcher-CD3 H chain)
  • Fifth protein a protein comprising a variable domain, a SnoopTag (binding partner), and a light chain of a Herceptin antibody (VHH-SnoopTag-Herceptin L chain)
  • Sixth protein a protein containing SnoopCatcher (binding partner) and the heavy chain and variable domain of the Herceptin antibody (VHH-SnoopCatcher-Herceptin H chain)
  • a plasmid vector capable of expressing VHH-SnoopTag-Herceptin L chain was constructed by the following procedure. First, the full length of a synthetic gene (Eurofins Genomics) containing a base sequence encoding a signal peptide, aGFP4 (single-chain antibody (soluble domain), SEQ ID NO: 9), G1 linker, SnoopTag (SEQ ID NO: 4), G2 linker, thrombin cleavage sequence, H4D5 light chain variable region, and H4D5 light chain constant region was amplified by PCR. The resulting full length synthetic gene was linked to an expression vector (pCAGEN) for animal cells to construct an expression vector for the recombinant protein.
  • pCAGEN expression vector
  • the expression vector contains the VHH-SnoopTag-Herceptin L chain region (SEQ ID NO: 15) in the following order from the N-terminus to the C-terminus: signal peptide, aGFP4, G1 linker, SnoopTag, G2 linker, thrombin cleavage sequence, H4D5 light chain variable region, and H4D5 light chain constant region, as shown in parentheses.
  • VHH-SnoopTag-Herceptin L chain region (SEQ ID NO: 15) [METPAQLLFLLLWLPESTG][QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS][GGSGG][KLGDIEFIKVNK][GGSGGGGSGG][LVPRGSHMHM][DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI][KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
  • a plasmid vector capable of expressing VHH-SnoopCatcher-Herceptin H chain was constructed by the following procedure. First, the full length of a synthetic gene (Eurofins Genomics) containing base sequences encoding the signal peptide, Ia1 (sequence number 11), G1 linker, SnoopCatcher (sequence number 3), G2 linker, thrombin cleavage sequence, H4D5 heavy chain variable region, H4D5 heavy chain constant region, hinge region, and Fc region was amplified by PCR. The resulting full length synthetic gene was linked to an animal cell expression vector (pCAGGS) to construct an expression vector for the recombinant protein.
  • pCAGGS animal cell expression vector
  • the expression vector is a VHH-SnoopCatcher-Herceptin H chain region (SEQ ID NO: 16) in which, from the N-terminus to the C-terminus, as shown in parentheses, the signal peptide, Ia1, G1 linker, SnoopCatcher, G2 linker, thrombin cleavage sequence, H4D5 heavy chain variable region, H4D5 heavy chain constant region, hinge region, and Fc region are linked in this order.
  • VHH-SnoopCatcher-Herceptin H chain region (SEQ ID NO: 16) [MEFGLSWLFLVAILKGVQ][CQVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GGSGG][KPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNDKHYITNEPIPPK][GGSGGGGSGG][LVPRGSHMHM][EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY L
  • Example 1 (1) transfection of the four plasmid expression vectors of the recombinant proteins, cell culture, and collection of the culture medium supernatant were performed in the same manner as in Example 1 (1).
  • the proteins were purified in the same manner as in Example 1 (2) to obtain purified samples.
  • the formation of complexes containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain, and complexes containing VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain was examined using SDS-PAGE. Specifically, 10 ⁇ l of 5 ⁇ SDS buffer was added to the purified sample (40 ⁇ l) and the sample was suspended.
  • Figure 9 is a photograph showing the results of SDS-PAGE.
  • the left side of the photograph shows molecular weight (kDa).
  • bands of the estimated molecular weights of the two complexes were detected in the purified sample.
  • Example 1 (4) the same method as in Example 1 (4) was used, except that a complex containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain as well as a complex containing VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain were used as samples. These results are shown in FIG. 10.
  • FIG. 10 is a photograph showing the results of SDS-PAGE.
  • the left side of the photograph shows the molecular weight (kDa).
  • bands of the estimated molecular weights of the complexes containing VHH-SpyTag-CD3 L chain and VHH-SpyCatcher-CD3 H chain, and the complexes containing VHH-SnoopTag-Herceptin L chain and VHH-SnoopCatcher-Herceptin H chain were not detected. Also, as shown in FIG.
  • a method for producing a heteromultimeric protein comprising the steps of: A complex formation step in which two proteins are contacted to form a first complex of the two proteins,
  • the two proteins include a first protein and a second protein, the first protein comprises, in order from N-terminus to C-terminus, a first binding tag, a first cleavage domain, and a first domain;
  • the second protein comprises, in order from N-terminus to C-terminus, a first binding partner capable of binding to a first binding tag, a second cleavage domain, and a second domain;
  • the first protein and the second protein are capable of forming a dimer by binding between the first domain and the second domain; forming the first complex by binding between the first binding tag and the first binding partner and between the first domain and the second domain;
  • a production method comprising
  • (Appendix 5) A manufacturing method described in any of appendix 1 to 4, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.
  • (Appendix 6) The method according to any one of claims 1 to 5, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the group consisting of the following (1) to (3): (1) a modified Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Modified Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag) (3) A modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 565-587 capable of binding to the Cpe0147 439-563 .
  • the complex formation step is a step of contacting four proteins to form a second complex of the four proteins, the four proteins include the first protein, the second protein, a third protein, and a fourth protein; the third protein comprises, in order from N-terminus to C-terminus, a second binding tag, a third cleavage domain, and a third domain; the fourth protein comprises, in order from N-terminus to C-terminus, a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain; the second protein and the fourth protein are capable of forming a dimer by binding between the second domain and the fourth domain; the third protein and the fourth protein are capable of forming a dimer by binding between the third domain and the fourth domain; forming the second complex by binding between the first binding tag and the first binding partner, between the second binding tag and the second binding partner, between the first domain and the second domain, between the second domain and the fourth domain, and between the third domain and the fourth domain; The method according to any
  • the second protein further comprises, at its C-terminus, a fifth cleavage domain and a third binding tag, in that order; the fourth protein further comprises, at its C-terminus, a sixth cleavage domain and a third binding partner capable of binding to the third binding tag, in that order;
  • the complex formation step further comprises binding the third binding tag to the third binding partner, The method of any one of appendixes 11 to 24, wherein in the generating step, the fifth cleavage domain and the sixth cleavage domain in the second complex are cleaved.
  • Appendix 26 26.
  • Appendix 27 27.
  • the method of claim 25 or 26, wherein the fifth cleavage domain and/or the sixth cleavage domain comprises a self-cleaving peptide and/or a cleavage sequence for a protease or peptidase.
  • Appendix 28 The method for production described in Appendix 27, wherein the protease cleavage sequence is a thrombin cleavage sequence.
  • Appendix 29 29.
  • the method of any one of claims 25 to 28, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.
  • Appendix 30 30.
  • the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the group consisting of (1) to (3) below: (1) a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag) (3) A modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 565-587 capable of binding to the Cpe0147 439-563 .
  • Appendix 33 The production method according to any one of Appendices 11 to 32, further comprising a first expression step of expressing the first protein, the second protein, the third protein, and the fourth protein in a host cell prior to the complex formation step.
  • Appendix 34 The method of any one of claims 11 to 33, further comprising a first purification step of purifying the second complex after the complex formation step.
  • Appendix 35 The method of any one of appendixes 11 to 34, further comprising a second purification step of purifying the heterodimer after the production step.
  • a protein comprising, in order from N-terminus to C-terminus, a first binding tag capable of binding to a first binding partner, a first cleavage domain, and a first domain.
  • Appendix 37 37.
  • the protein of claim 36, wherein the first cleavage domain comprises a self-cleaving peptide and/or a cleavage sequence for a protease or peptidase.
  • Appendix 38 38.
  • the protein according to claim 37, wherein the protease cleavage sequence is a thrombin cleavage sequence.
  • Appendix 39 39.
  • (Appendix 42) A protein comprising, in order from N-terminus to C-terminus, a first binding partner capable of binding to a first binding tag, a second cleavage domain, and a second domain.
  • the protein of claim 42, wherein the second cleavage domain comprises a self-cleaving peptide and/or a protease or peptidase cleavage sequence.
  • (Appendix 45) 45 The protein of any one of claims 42 to 44, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond.
  • (Appendix 46) 46 The protein according to any one of claims 42 to 45, wherein the first binding tag and the first binding partner are a combination of a binding tag and a binding partner selected from the group consisting of (1) to (3) below: (1) a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag) (3) A modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 565-587 capable of binding to the Cpe0147 439-563 .
  • (Appendix 47) 47 The protein of any one of claims 42 to 46, wherein the second domain is a heavy chain of an antibody that binds to a first target.
  • (Appendix 48) 48 The protein of any of claims 42 to 47, further comprising at the C-terminus, a fifth cleavage domain, and a third binding tag or a third binding partner capable of binding to the third binding tag, in that order.
  • the protein of claim 48, wherein the fifth cleavage domain comprises a self-cleaving peptide and/or a protease or peptidase cleavage sequence.
  • (Appendix 50) 50 50.
  • the protein of any one of claims 48 to 51, wherein the second cleavage domain and the fifth cleavage domain are the same cleavage domain.
  • Appendix 53 53.
  • the protein of any of claims 48-52, wherein the first binding tag and first binding partner, and the third binding tag and third binding partner are different binding tag and binding partner combinations.
  • the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the group consisting of (1) to (3) below: (1) a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag) (3) A modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 565-587 capable of binding to the Cpe0147 439-563 .
  • (Appendix 55) The protein comprising, in order from N-terminus to C-terminus, a second binding tag capable of binding to a second binding partner, a third cleavage domain, and a third domain.
  • (Appendix 56) 56.
  • (Appendix 60) 60 The protein of any of claims 55 to 59, wherein the third domain is a light chain of an antibody that binds to a second target.
  • (Appendix 61) A protein comprising, in order from N-terminus to C-terminus, a first binding partner capable of binding to a second binding tag, a fourth cleavage domain, and a fourth domain.
  • the protein of claim 62 wherein the protease cleavage sequence is a thrombin cleavage sequence.
  • (Appendix 66) 66 The protein of any one of claims 61 to 65, wherein the fourth domain is a heavy chain of an antibody that binds to a second target.
  • the protein of claim 67, wherein the sixth cleavage domain comprises a self-cleaving peptide and/or a protease or peptidase cleavage sequence.
  • the protein of any of claims 67 to 70, wherein the fourth cleavage domain and the sixth cleavage domain are the same cleavage domain.
  • (Appendix 72) 72 The protein of any of claims 67 to 71, wherein the second binding tag and second binding partner, and the third binding tag and third binding partner are different binding tag and binding partner combinations.
  • (Appendix 73) 73 The protein according to any one of claims 67 to 72, wherein the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the group consisting of (1) to (3) below: (1) a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag) (3) A modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 565-587 capable of binding to the Cpe0147 439-563 .
  • Appendix 74 A protein according to any one of appendices 36 to 73 for use in a method for producing a heteromultimeric protein according to any one of appendices 1 to 35.
  • ⁇ Heteromultimeric proteins> It contains two proteins, The two proteins include a first protein and a second protein, the first protein comprises, in order from N-terminus to C-terminus, a first binding tag, a first cleavage domain, and a first domain; the second protein comprises, in order from N-terminus to C-terminus, a first binding partner capable of binding to the first binding tag, a second cleavage domain, and a second domain; the first protein and the second protein form a dimer by binding between the first domain and the second domain; A protein having said first binding tag and said first binding partner bound thereto.
  • (Appendix 76) 76 The protein of claim 75, wherein the first cleavage domain and the second cleavage domain are the same cleavage domain. (Appendix 77) 77. The protein of claim 75 or 76, wherein the first cleavage domain and/or the second cleavage domain comprise a self-cleaving peptide and/or a protease or peptidase cleavage sequence. (Appendix 78) 78. The protein of any one of claims 75 to 77, wherein the first binding tag and the first binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond. (Appendix 79) 79.
  • the bond between the first domain and the second domain is a disulfide bond.
  • Appendix 81 Further comprising a third protein and a fourth protein, the third protein comprises, in order from N-terminus to C-terminus, a second binding tag, a third cleavage domain, and a third domain; the fourth protein comprises, in order from N-terminus to C-terminus, a second binding partner capable of binding to the second binding tag, a fourth cleavage domain, and a fourth domain; the second protein and the fourth protein form a dimer by binding between the second domain and the fourth domain; the third protein and the fourth protein form a dimer by binding between the third domain and the fourth domain; the first binding tag and the first binding partner bind; 81.
  • Appendix 86 86.
  • (Appendix 87) 87 The protein of any one of claims 81 to 86, wherein the bond between the third domain and the fourth domain and/or the bond between the second domain and the fourth domain is a disulfide bond.
  • the first domain is a light chain of an antibody that binds to a first target;
  • the second domain is a heavy chain of an antibody that binds to the first target; said third domain being a light chain of an antibody that binds to a second target; 88.
  • the protein of claim 88 wherein the antibody that binds to the first target and the antibody that binds to the second target recognize different epitopes.
  • Appendix 90 90.
  • the protein of claim 88 or 89 wherein the antibody that binds to the first target and the antibody that binds to the second target recognize different antigens.
  • Appendix 91 91.
  • the protein of any of claims 88 to 90, wherein the antibody that binds to the first target and the antibody that binds to the second target are IgG, IgA, IgE, IgD, or IgM.
  • Appendix 92 92.
  • the protein of claim 91 wherein the IgG is IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgG4.
  • (Appendix 93) 93 The protein of any of claims 81 to 92, wherein the first cleavage domain, the second cleavage domain, the third cleavage domain, and the fourth cleavage domain are the same cleavage domain.
  • the protein of any of claims 81 to 93 wherein the first binding tag and first binding partner, and the second binding tag and second binding partner are different binding tag and binding partner combinations.
  • the second protein further comprises, at its C-terminus, a fifth cleavage domain and a third binding tag, in that order; the fourth protein further comprises, at its C-terminus, a sixth cleavage domain and a third binding partner capable of binding to the third binding tag, in that order; 95.
  • Appendix 97 97.
  • Appendix 98 98.
  • Appendix 99 99.
  • the protein of any of claims 95 to 98, wherein the third binding tag and the third binding partner are a peptide tag and a peptide capable of spontaneously forming a covalent bond. (Appendix 100) 99.
  • the third binding tag and the third binding partner are a combination of a binding tag and a binding partner selected from the group consisting of: (1) a Streptococcus pyogenes surface protein (SpyCatcher) and a peptide tag (SpyTag) capable of binding to the SpyCatcher; (2) Streptococcus pneumoniae protein (SnoopCatcher) and a peptide tag capable of binding to the SnoopCatcher (SnoopTag) (3) A modified Clostridium perfringens protein Cpe0147 439-563 and a peptide tag Cpe0147 565-587 capable of binding to the Cpe0147 439-563 . (Appendix 101) 101.
  • ⁇ Transformants> (Appendix 105) A transformant comprising the nucleic acid described in Appendix 103 and/or the expression vector described in Appendix 104.
  • ⁇ Protein production method> (Appendix 106) A method for producing a protein, comprising an expression step of expressing the nucleic acid described in Appendix 103 and/or the expression vector described in Appendix 104.
  • the expression step comprises: A culturing step of culturing the transformant according to claim 105; isolating the protein according to any one of claims 36 to 102; The method of claim 106, comprising:
  • heteromultimeric proteins such as bispecific antibodies can be efficiently produced. Therefore, the present disclosure is extremely useful, for example, in the fields of medicine and pharmaceutical manufacturing.

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Abstract

L'invention concerne un nouveau procédé de production d'un hétéro-corps qui permet la production d'une protéine hétéromultimère telle qu'un anticorps bispécifique.　Le procédé de production de la présente divulgation, qui consiste à produire une protéine hétéromultimérique, comprend les deux étapes suivantes. Une première étape de formation de complexe pour mettre en contact deux protéines afin de constituer un premier complexe des deux protéines, dans laquelle : les deux protéines comprennent une première protéine et une deuxième protéine ; la première protéine comprend, de l'extrémité N à l'extrémité C, une première marqueur de liaison, un premier domaine de clivage et un premier domaine, dans cet ordre ; la deuxième protéine comprend, de l'extrémité N à l'extrémité C, un premier partenaire de liaison capable de se lier au premier marqueur de liaison, un deuxième domaine de clivage et un deuxième domaine, dans cet ordre ; la première protéine et la deuxième protéine sont capables de constituer un dimère par liaison entre le premier domaine et le deuxième domaine ; et le premier marqueur de liaison se lie au premier partenaire de liaison et le premier domaine se lie au deuxième domaine pour former le premier complexe. Une étape de génération pour cliver le premier domaine de clivage et le deuxième domaine de clivage dans le premier complexe afin de générer un hétérodimère du premier domaine et du deuxième domaine.
PCT/JP2023/034748 2022-09-30 2023-09-25 Procédé de production de protéine hétéromultimère, protéine, acide nucléique, vecteur d'expression, transformant et procédé de production de protéine WO2024071043A1 (fr)

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WO2012071649A1 (fr) * 2010-11-29 2012-06-07 National Research Council Of Canada Agents bivalents de liaison dimérisés par liaison covalente
WO2018213335A1 (fr) * 2017-05-16 2018-11-22 Scalmibio, Inc. Anticorps activables et leurs procédés d'utilisation
JP2019535321A (ja) * 2016-11-16 2019-12-12 オークランド ユニサービシーズ リミティド タンパク質ライゲーションの方法及びその使用
WO2021173612A1 (fr) * 2020-02-26 2021-09-02 Sorrento Therapeutics, Inc. Protéines de liaison à un antigène activables avec des fractions de masquage universelles
JP2022525777A (ja) * 2019-03-18 2022-05-19 バイオ－ラッド エービーディー セロテック ゲーエムベーハー 抗原結合タンパク質

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WO2012071649A1 (fr) * 2010-11-29 2012-06-07 National Research Council Of Canada Agents bivalents de liaison dimérisés par liaison covalente
JP2019535321A (ja) * 2016-11-16 2019-12-12 オークランド ユニサービシーズ リミティド タンパク質ライゲーションの方法及びその使用
WO2018213335A1 (fr) * 2017-05-16 2018-11-22 Scalmibio, Inc. Anticorps activables et leurs procédés d'utilisation
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